Microfabricated flowthrough porous apparatus for discrete detection binding reactions

ABSTRACT

An improved apparatus for conducting a multiplicity of individual and simultaneous binding reactions is described. The apparatus comprises a substrate on which are located discrete and isolated sites for binding reactions. The apparatus is characterized by discrete and isolated regions that extend through a substrate and terminate on a second surface thereof, such that when a test sample is applied to the substrate, it is capable of penetrating through each such region during the course of the binding reaction. The apparatus is especially useful for sequencing by hybridization of DNA molecules.

This application is a continuation of U.S. Ser. No. 09/063,356, filedApr. 21, 1998, now U.S. Pat. No. 6,893,816; which is a continuation ofU.S. Ser. No. 08/631,751, filed Apr. 10, 1996, now U.S. Pat. No.5,843,767; which is a continuation of PCT/US94/12282, filed Oct. 27,1994; which is a continuation-in-part of U.S. Ser. No. 08/141,969, filedOct. 28, 1993, now abandoned.

BACKGROUND OF THE INVENTION

Several forms of arrayed hybridization reactions are currently beingdeveloped under the common rubric of “sequencing by hybridization”(SBH). Included are “format 1” versions of SBH, involving stepwisehybridization of different oligonucleotide probes with arrays of DNAsamples gridded onto membranes, and “format 2” implementations,involving hybridization of a single nucleic acid “target sequence” to anarray of oligonucleotide probes tethered to a flat surface orimmobilized within a thin gel matrix. The term “genosensor” hasheretofore referred to a form of SBH in which oligonucleotides aretethered to a surface in a two-dimensional array and serve asrecognition elements for complementary sequences present in a nucleicacid “target” sequence. The genosensor concept further includesmicrofabricated devices in which microelectronic components are presentin each test site, permitting rapid, addressable detection ofhybridization across the array.

The present invention provides a novel flow-through genosensor, in whichnucleic acid recognition elements are immobilized within densely packedpores or channels, arranged in patches across a wafer of solid supportmaterial. Known microfabrication techniques are available for producingmicrochannel or nanochannel glass and porous silicon useful as supportwafers. Flow-through genosensors utilize a variety of conventionaldetection methods, including microfabricated optical and electronicdetection components, film, charge-coupled-device arrays, camera systemsand phosphor storage technology.

The following advantages for the novel flow-through apparatus herein ascompared to known flat surface designs are obtained:

(1) improved detection sensitivity due to the vastly increased surfacearea which increases the quantity of nucleic acid bound per crosssectional area;

(2) minimization of a rate-limiting diffusion step preceding thehybridization reaction (reducing the time required for the averagetarget molecule to encounter a surface-tethered probe from hours tomilliseconds), speeding hybridization and enabling mismatchdiscrimination at both forward and reverse reactions;

(3) enablement of the analysis of dilute nucleic acid solutions becauseof the ability to gradually flow the solution through the porous wafer;

(4) facilitation of subsequent rounds of hybridization involvingdelivery of probes to specific sites within the hybridization array;

(5) facilitation of the recovery of bound nucleic acids from specifichybridization sites within the array, enabling the further analysis ofsuch recovered molecules; and

(6) facilitation of the chemical bonding of probe molecules to thesurface within each isolated region due to the avoidance of the rapiddrying of small droploets of probe solution on flat surfaces exposed tothe atmosphere.

Accordingly, the present invention provides an improved apparatus andmethod

-   -   for the simultaneous conduct of a multiplicity binding reactions        on a substrate,    -   which substrate is a microfabricated device comprising a set of        discrete and isolated regions on the substrate,    -   such that each such discrete and isolated region corresponds to        the location of one such binding reaction,    -   in which each such discrete and isolated region contains an        essentially homogeneous sample of a biomolecule of discrete        chemical structure fixed to such bounded region,    -   such that upon contact between the substrate and a sample        (hereinafter, “test sample”) containing one or more molecular        species capable of controllably binding with one or more of the        pre-determined biomolecules,    -   the detection of the bounded regions in which such binding has        taken place yields a pattern of binding capable of        characterizing or otherwise identifying the molecular species in        the test sample.

The present invention specifically provides novel high-density andultra-high density microfabricated, porous devices for the conductionand detection of binding reactions. In particular, the present inventionprovides improved “genosenors” and methods for the use thereof in theidentification or characterization of nucleic acid sequences throughnucleic acid probe hybridization with samples containing anuncharacterized polynucleic acid, e.g., a cDNA, mRNA, recombinant DNA,polymerase chain reaction (PRC) fragments or the like, as well as otherbiomolecules.

During the past decade microfabrication technology has revolutionizedthe electronics industry and has enabled miniaturization and automationof manufacturing processes in numerous industries. The impact ofmicrofabrication technology in biomedical research can be seen in thegrowing presence of microprocessor-controlled analytical instrumentationand robotics in the laboratory, which is particularly evident inlaboratories engaged in high throughput genome mapping and sequencing.The Human Genome Project is a prime example of a task that whoseeconomics would greatly benefit from microfabricated high-density andultra-high density hybridization devices that can be broadly applied ingenome mapping and sequencing.

Hybridization of membrane-immobilized DNAs with labeled DNA probes is awidely used analytical procedure in genome mapping. Robotic devicescurrently enable gridding of 10,000–15,000 different target DNAs onto a12 cm×8 cm membrane. Drmanac, R., Drmanac, S., Jarvis, J. and Labat, 1.1993. in Venter, J. C. (Ed.), Automated DNA Sequencing and AnalysisTechniques, Academic Press, in press, and Meier-Ewert, S., Maier, E.,Ahmadi, A., Curtis, J. and Lehrach, H. 1993. Science 361:375–376.Hybridization of DNA probes to such filters has numerous applications ingenome mapping, including generation of linearly ordered libraries,mapping of cloned genomic segments to specific chromosomes or megaYACs,cross connection of cloned sequences in cDNA and genomic libraries, etc.Recent initiatives in “sequencing by hybridization” (SBH) aim towardminiaturized, high density hybridization arrays. A serious limitation tominiaturization of hybridization arrays in membranes or on flat surfacesis the quantity of DNA present per unit cross sectional area, which (ona two-dimensional surface) is a function of the surface area. Thisparameter governs the yield of hybridized DNA and thus the detectionsensitivity.

Genosensors, or miniaturized “DNA chips” are currently being developedin several laboratories for hybridization analysis of DNA samples. DNAchips typically employ arrays of DNA probes tethered to flat surfaces,e.g., Fodor, S. P. A., Read, J. L., Pirrung, M. C., Stryer, L., Lu, A.T. and Solas, D. 1991. Science 251:767–773, Southern, E. M., Maskos, U.and Elder, J. K. 1992. Genomics 13:1008–1017, Eggers, M. D., Hogan, M.E., Reigh, R. K., Lamture, J. B., Beattie, K. L., Hollis, M. A.,Ehrlich, D. J., Kosicki, B. B., Shumaker, J. M., Varma, R. S., Burke, B.E., Murphy, A. and Rathman, D. D. 1993. Advances in DNA SequencingTechnology, SPIE Conference, Los Angeles, Calif., and Beattie, K.,Eggers, M., Shumaker, J., Hogan, M., Varma, R., Lamture, J., Hollis, M.,Ehrlich, D. and Rathman, D. 1993. Clin. Chem. 39:719–722, to acquire ahybridization pattern that reflects the nucleotide sequence of thetarget DNA. The detection limit for hybridization on flat-surfacegenosensors, as in membrane hybridization, is limited by the quantity ofDNA that can be bound to a two dimensional area

Another limitation of these prior art approaches is the fact that a flatsurface design introduces a rate-limiting step in the hybridizationreaction, i.e., diffusion of target molecules over relatively longdistances before encountering the complementary probes on the surface.In contrast, the microfabricated apparatus according to the presentinvention is designed to overcome the inherent limitations in currentsolid phase hybridization materials, eliminating the diffusion-limitedstep in flat surface hybridizations and increasing the cross sectionaldensity of DNA.

Typically microfabricated genosensor devices are characterized by acompact physical size and the density of components located therein.Known microfabricated binding devices are typically rectangularwafer-type apparatuses with a surface area of approximate one cm², e.g.,1 cm×1 cm. The bounded regions on such devices are typically present ina density of 10²–10⁴ regions/cm², although the desirability ofconstructing apparatuses with much higher densities has been regarded asan important objective. See Eggers and Beattie, cited above, fordiscussion of strategies for the construction of devices with higherdensities for the bounded regions.

The microfabricated apparatuses as described herein are known to beuseful for a variety of analytical tasks, including nucleic acidsequence analysis by hybridization (SBH), analysis of patterns of geneexpression by hybridization of cellular mRNA to an array ofgene-specific probes, immunochemical analysis of protein mixtures,epitope mapping, assay of receptor-ligand interactions, and profiling ofcellular populations involving binding of cell surface molecules tospecific ligands or receptors immobilized within individual bindingsites. Although nucleic acid analysis is one principal use for such anmicroapparatus, it is advantageously applied to a broad range ofmolecular binding reactions involving small molecules, macromolecules,particles, and cellular systems. See, for example, the uses described inPCT Published Application WO 89/10977.

Ordinarily the microfabricated apparatus is used in conjunction with aknown detection technology particularly adapted to discriminatingbetween bounded regions in which binding has taken place and those inwhich no binding has occurred and for quantitating the relative extentof binding in different bounded regions. In DNA and RNA sequencedetection, autoradiography and optical detection are advantageouslyused. Autoradiography is performed using ³²P or ³⁵S labelled samples.For traditional DNA sequence analysis applications, nucleic acidfragments are end-labeled with ³²P and these end-labeled fragments areseparated by size and then placed adjacent to x-ray film as needed toexpose the film, a function of the amount of radioactivity adjacent to aregion of film. Alternatively, phosphorimager detection methods may beused.

Optical detection of fluorescent-labelled receptors is also employed indetection. In traditional sequencing, a DNA base-specific fluorescentdye is attached covalently to the oligonucleotide primers or to thechain-terminating dideoxynucleotides used in conjunction with DNApolymerase. The appropriate absorption wavelength for each dye is chosenand used to excite the dye. If the absorption spectra of the dyes areclose to each other, a specific wavelength can be chosen to excite theentire set of dyes. One particularly useful optical detection techniqueinvolves the use of ethidium bromide, which stains duplex nucleic acids.The fluorescence of these dyes exhibits an approximate twenty-foldincrease when it is bound to duplexed DNA or RNA, when compared to thefluorescence exhibited by unbound dye or dye bound to single-strandedDNA. This dye is advantageously used to detect the presence ofhybridized polynucleic acids.

A highly preferred method of detection is a charge-coupled-device arrayor CCD array. With the CCD array, a individual pixel or group of pixelswithin the CCD array is placed adjacent to each confined region of thesubstrate where detection is to be undertaken. Light attenuation, causedby the greater absorption of an illuminating light in test sites withhybridized molecules, is used to determine the sites where hybridizationhas taken place. Lens-based CCD cameras can also be used.

Alternatively, a detection apparatus can be constructed such thatsensing of changes in AC conductance or the dissipation of a capacitorplaced contiguous to each confined region can be measured. Similarly, byforming a transmission line between two electrodes contiguous to eachconfined region hybridized molecules can be measured by theradio-frequence (RF) loss. The preferred methods for use herein aredescribed in, Optical and Electrical Methods and Apparatus for MoleculeDetection, PCT Published Application WO 93/22678, published Nov. 11,1993, and expressly incorporated herein by reference.

Methods for attaching samples of substantially homogeneous biomoleculesof a pre-determined structure to the confined regions of themicroapparatus are likewise known. One preferred method of doing so isto attach these biomolecules covalently to surfaces such as glass orgold films. For example, methods for attachments of oligonucleotideprobes to glass surfaces are known. A primary amine is introduced at oneterminus during the chemical synthesis thereof. Optionally, one or moretriethylene glycol units may be introduced therebetween as spacer units.After derivatizing the glass surface in the confined region withepoxysilane, the primary amine terminus of the oligonucleotide can becovalently attached thereto.

See Beattie, et al., cited above, for a further description of thistechnology for fixing the pre-determined biomolecules in the boundedregions of the microfabricated apparatus.

RELATED ART

Khrapko, K. R., et al., A method for DNA sequencing by hybridizationwith oligonucleotide matrix, J. DNA Sequencing and Mapping, 1:375–388(1991), Drmanac, Radoje, et al., Sequencing by hybridization: Towards anautomated sequencing of one million M13 clones arrayed on membranesElectrophoresis 13:566–573 (1992), Meier-Ewert, Sebastian, An automatedapproach to generating expressed sequence catalogues, Nature 361:375–376(1993), Drmanac, R., et al., DNA Sequence Determination byHybridization: A Strategy for Efficient Large-Scale Sequencing, Science260:1649–1652 (1993), Southern, E. M., et al., Analyzing and ComparingNucleic Acid Sequences by Hybridization to Arrays of Oligonucleotides:Evaluation Using Experimental Models, Genomics 13:1008–1017 (1992), andSaiki, Randall K., et al., Genetic analysis of amplified DNA withimmobilized sequence-specific oligonucleotide probes, Proc. Natl. Acad.Sci. USA 86:6230–6234 (1989) describe sequence-by-hybridizationdeterminations, including via the use of arrays of oligonucleotidesattached to a matrix or substrate. Eggers, Mitchell D., et al.,Genosensors: microfabricated devices for automated DNA sequenceanalysis, SPIE Proceedings Series, Advances in DNA Sequence Technology,Proceedings Preprint, The International Society for Optical Engineering,21 Jan. 1993; Beattie, Kenneth, et al., Genosensor Technology, ClinicalChemistry 39:719–722 (1993); Lamture, J. B., et al., Direct detection ofnucleic acid hybridization on the surface of a charge coupled device,Nucl. Acids Res. 22:2121–2124 (1994); and Eggers, M., et al., Amicrochip for quantitative detection of molecules utilizing luminescentand radioisotope reporter groups, Biotechniques 17:516–525 (1994)describe the general strategies and methodologies for designingmicrofabricated devices useful in sequencing by hybridization (SBH) forDNA.

SUMMARY OF THE INVENTION

The present invention particularly provides:

(a) In a microfabricated device comprising—

(1) a substrate containing a multiplicity of discrete and isolatedregions arrayed across a surface thereof and adapted to interact with orintegrally interacting with a detecting means capable of identifying andaddressing each of said regions and determining and reporting whether abinding reaction has taken place therein, and

(2) essentially homogeneous samples of biomolecules of pre-determinedstructures fixed in each of said discrete and isolated regions, suchthat the detection of a binding reaction between said biomolecules inone or more of said regions and a test sample provides informationcapable of identifying or otherwise characterizing the molecular speciesin said test sample,

the improvement that comprises:

discrete and isolated regions that extend through said substrate andterminate on a second surface thereof such that said test sample uponcontact with said substrate is capable of penetrating therethroughduring the course of said binding reaction.

(b) The improvement above wherein said biomolecules are oligonucleotidesand said test sample comprises polynucleic acids.

(c) The improvement above wherein said substrate is a nanoporous glasswafer.

(d) The improvement above wherein said discrete and isolated regionscomprise tapered conical wells bonded to one face of said nanoporousglass wafer.

(e) The improvement above comprising a high density array, wherein eachof said discrete and isolated regions on said nanoporous glass wafer hasa largest diameter of about 100 μm, the spacing between adjacent regionsis about 500 μm, said wafer is about 100 μm in thickness, whereby thevolume of said region within the wafer is about 40 nL and the density ofsaid regions on said wafer is about 400 regions/cm².

(f) The improvement above comprising an ultra-high density array,wherein each of said discrete and isolated regions on said nanoporousglass wafer has a largest diameter of about 50 μm, the spacing betweenadjacent regions is about 150 μm, said wafer is about 50 μm inthickness, whereby the volume of said region within the wafer is aboutone nL and the density of said regions on said wafer is about 4,400regions/cm².

(g) The improvement above comprising an array, wherein each of saiddiscrete and isolated regions on said nanoporous glass wafer has alargest diameter of from about 5 μm to about 2000 μm, the spacingbetween adjacent regions is from about 0.1 to 10 times said largestdiameter, and said wafer is from about 10 μm to about 500 μm inthickness.

(h) The improvement above wherein the contact between said test sampleand said discrete and isolated regions is by flooding the first surfaceof said substrate with said test sample and placing said second surfaceof said substrate under negative pressure relative to said firstsurface, whereby the resulting vacuum facilitates the flow through saidsubstrate.

(i) The improvement above wherein said oligonucleotides are fixed insaid isolated and discrete regions on said substrate by attaching aterminal primary amine derivative of said oligonucleotide to a glasssubstrate derivatized with epoxysilane.

(j) The improvement above wherein said oligonucleotide-silane fixationcomprises the incorporation of one or more triethylene glycol phosphorylunits, whereby optimal spacing between said glass surface and the basepairs of said oligonucleotide is achieved.

(k) The improvement above wherein said oligonucleotides are fixed insaid isolated and discrete regions on said substrate by attaching aterminal bromoacetylated amine derivative of said oligonucleotide to aplatinum or gold substrate derivatized with a dithioalkane.

(l) The improvement above wherein said detection of said bindingreaction is detection by a charge-coupled device (CCD) employed todetect hybridization of radioisotope-, fluorescent-, orchemiluminescent-labelled polynucleic acids.

(m) A microfabricated device for simultaneously conducting amultiplicity of binding reactions, comprising:

(1) a substrate providing a rigid support for said device;

(2) an array of discrete and isolated regions arranged across a surfaceof said substrate and extending therethrough to a second surface of saidsubstrate, thereby forming pores in said substrate;

(3) substantially homogeneous samples of a pre-determined set ofbiomolecules, each such sample being fixed in one or more of saidregions, such that one or more of said biomolecules is capable ofbinding with a molecular species in a test sample passing therethrough;and

(4) a detection means capable of determining for each such regionwhether a binding reaction has taken place and reporting the resultthereof.

(n) A device as described above further comprising a means for providingfluidic flow through the substrate.

(o) In a method for using a microfabricated device for theidentification of the molecular species contained in a test sample, saiddevice comprising—

(1) a substrate containing a multiplicity of discrete and isolatedregions arrayed across a surface thereof and adapted to interact with orintegrally interacting with a detecting means capable of characterizingor otherwise identifying and addressing each of said regions anddetermining and reporting whether a binding reaction has taken placetherein, and

(2) essentially homogeneous samples of biomolecules of pre-determinedstructures fixed in each of said discrete and isolated regions, suchthat the detection of a binding reaction between said biomolecules inone or more of said regions and said test sample provides informationcapable of characterizing or otherwise identifying the molecular speciesin said test sample,

the improvement that comprises:

allowing said test sample, during the course of said binding reaction,to penetrate through said discrete and isolated regions by constructingsaid regions to contain pores that extend through said substrate andterminate on a second surface thereof.

The devices of the present invention are used to characterize orotherwise identify molecular species capable of controllably binding tobiomolecules in the same manner and using the same binding regimens asare known in the art. Although uses of these novel devices includeantibody-antigen and ligand-receptor binding, a major use of the presentinvention is in the field of nucleic acid sequence analysis. Twofundamental properties of DNA are vital to its coding and replicationalfunctions in the cell.

(1) The arrangement of “bases” [adenenine (A), guanine (G), cytosine (C)and thymine (T)] in a specific sequence along the DNA chain defines thegenetic makeup of an individual. DNA sequence differences account forthe differences in physical characteristics between species and betweendifferent individuals of a given species

(2) One strand of DNA can specifically pair with another DNA strand toform a double-stranded structure in which the bases are paired byspecific hydrogen bonding: A pairs with T and G pairs with C. Specificpairing also occurs between DNA and another nucleic acid, ribonucleicacid (RNA), wherein uracil (U) in RNA exhibits the same base pairingproperties as T in DNA.

The specific pattern of base pairing (A with T or U and G with C) isvital to the proper functioning of nucleic acids in cells, and alsocomprises a highly specific means for the analysis of nucleic acidsequences outside the cell. A nucleic acid strand of specific basesequence can be used as a sequence recognition element to “probe” forthe presence of the perfectly “complementary” sequence within a nucleicacid sample (Conner, et al., Proc. Natl. Acad. Sci., U.S.A., 80:278–282(1983)). Thus, if a sample of DNA or RNA is “annealed” or “hybridized”with a nucleic acid “probe” containing a specific base sequence, theprobe will bind to the nucleic acid “target” strand only if there isperfect (or near-perfect) sequence complementarity between probe andtarget. The hybridization event which indicates the presence of aspecific base sequence in a nucleic acid sample is typically detected byimmobilization of the nucleic acid sample or the probe on a surface,followed by capture of a “tag” (for example, radioactivity orfluorescence) carried by the complementary sequence.

DNA hybridization has been employed to probe for sequence identity ordifference between DNA samples, for example in the detection ofmutations within specific genetic regions (Kidd, et al., N. Engl. J.Med., 310:639–642 (1984); Saiki, et al., N. Engl. J. Med., 319:537–541(1988); Saiki, et al., Proc. Natl. Acad. Sci., U.S.A., 86:6230–6234(1989)). Although DNA probe analysis is a useful means for detection ofmutations associated with genetic diseases, the current methods arelimited by the necessity of performing a separate hybridization reactionfor detection of each mutation. Many human genetic diseases, forexample, cancer (Hollstein, et al., Science, 253:49–53 (1991)) areassociated with any of a large number of mutations distributed at manylocations within the affected genes. In these cases it has beennecessary to employ laborious DNA sequencing procedures to identifydisease-associated mutations. The problem is compounded when there is aneed to analyze a large number of DNA samples, involving populations ofindividuals. Detection of mutations induced by exposure to genotoxicchemicals or radiation is of interest in toxicology testing andpopulation screening, but again, laborious, costly and time consumingprocedures are currently necessary for such mutational analyses.

In addition to causing genetic diseases, mutations are also responsiblefor DNA sequence polymorphisms between individual members of apopulation. Genetic polymorphisms are DNA sequence changes at any givengenetic locus which are maintained in a significant fraction of theindividuals within a population. DNA sequence polymorphisms can serve asuseful markers in genetic mapping when the detectable DNA sequencechanges are closely linked to phenotypic markers and occur at afrequency of at least 5% of the individuals within a population. Inaddition, polymorphisms are employed in forensic identification andpaternity testing. Currently employed methods for detecting geneticpolymorphisms involve laborious searches for “restriction fragmentlength polymorphisms” (RFLPS) (Lander and Bottstein, Proc, Natl. Acad,Sci., U.S.A., 83:7353–7357 (1986)), the likewise laborious use of gelelectrophoretic DNA length analysis, combined with a DNA amplificationprocedure which utilizes oligonucleotide primers of arbitrary sequence(Williams, et al., Nucl. Acids Res., 18:6531–6535 (1991); Welsh andMcClelland, Nucl. Acids Res., 18:7213–7218 (1991)), and the gelelectrophoretic analysis of short tandem repeat sequences of variablelength) in genomic DNA. Weber, James L., Genomics 7: 524–530 (1990) andWeber, James L., Am. J. Hum. Genet. 44: 388–396 (1989).

Another kind of DNA sequence variation is that which occurs betweenspecies of organisms, which is of significance for several reasons.First, identification of sequence differences between species can assistin the determination of the molecular basis of phenotypic differencesbetween species. Second, a survey of sequence variation within aspecific gene among numerous related species can elucidate a spectrum ofallowable amino acid substitutions within the protein product encoded bythe gene, and this information is valuable in the determination ofstructure-function relationships and in protein engineering programs.However, this type of targeted DNA sequence comparison is extremelylaborious, time consuming and costly if carried out by current DNAsequencing methodology. Additionally, genetic sequence variation canform the basis of specific identification of organisms, for example,infectious micro-organisms.

The apparatus of the present invention is employed in a variety ofanalytical tasks, including nucleic acid sequence analysis byhybridization, analysis of patterns of gene expression by hybridizationof cellular mRNA to an array of gene-specific probes, immunochemicalanalysis of protein mixtures, epitope mapping, assay of receptor-ligandinteractions, and profiling of cellular populations involving binding ofcell surface molecules to specific ligands or receptors immobilizedwithin individual binding sites. Although nucleic acid analysis isspecifically taught in this disclosure, the present invention can beequally applied to a broad range of molecular binding reactionsinvolving small molecules, macromolecules, particles, and cellularsystems.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the use of an array of tapered sample wells that comprisea rigidifying support member for the porous wafer containing 0.1–10micron diameter channels comprising the bonding region for thebiomolecules fixed therein. As described below, the binding region is amicroporous or nanoporous glass wafer to which the upper polymeric layeris attached.

FIG. 2 depicts the packaging of a wafer substrate in a sealed lowerchamber to which a vacuum may be applied such that material applied toan upper reservoir contacts with the upper surface of the poroussubstrate is driven through the sample wells. Specifically depicted inFIG. 2 is the use of a Delrin O-Ring comprising the wafer-lower chamberseal.

FIG. 3 depicts a porous silicon wafer with integral sample wells.Procedures for constructing the depicted device are described in Example3.

FIG. 4 depicts the same vacuum-containing wafer-lower chamber apparatusof FIG. 2 with an additionally optional pressurized upper chamber.Again, as depicted, the upper chamber is sealed by use of a DelrinO-Ring.

FIG. 5 provides an idealized schematic depiction of the results of anhprt mutation detection assay on a device in accordance with the presentinvention. The sequence depicted on the left side of the figurecorresponds to nucleotides 23–55 of SEQ ID NO:2. One of the twosequences in the right side of the figure corresponds to nucleotides3–22 of SEQ ID NO:4 (sequence with A in the 16th position from the left)and the other to nucleotides 3–22 of SEQ ID NO:5 (bottom sequence with Greplacing A at position 16).

FIG. 6 provides an idealized schematic depiction of a hybridizationassay performed to profile gene expression under different experimentalconditions. Details of the assay procedure are provided in Example 11.

DETAILED DESCRIPTION

The present invention is more readily understood through the followingpreferred embodiments:

EXAMPLE 1 Nanochannel Glass (NCG) Wafers

Two types of nanochannel glass arrays developed at the Naval ResearchLaboratory are used as high surface area nanoporous support structuresto tether DNA targets or probes for hybridization. NCG materials areunique glass structures containing a regular geometric array of parallelholes or channels as small as 33 nm in diameter or as large as severalmicrometers in diameter. See Tonucci, R. J., Justus, B. L., Campillo, A.J. and Ford, C. E. 1992. Science 258:783–785. These nanochannel glassstructures can possess packing densities in excess of 3×10¹⁰ channelsper square centimeter, fabricated in various array configurations. Avariety of materials can be immobolized or fixed to the glass surfaceswithin the channels of the NCG array, to yield a high surface area tovolume ratio.

Nanochannel glass arrays are fabricated by arranging dissimilar glassesin a predetermined configuration, where at least one glass type isusually acid etchable. Construction of a two-dimensional hexagonal closepacking array begins by insertion of a cylindrical acid etchable glassrod (referred to as the channel glass) into an inert glass tube(referred to as the matrix glass) whose inner dimensions match that ofthe rod. The pair is then drawn under vacuum to reduce the overallcross-section to that of a fine filament. The filaments are thenstacked, re-fused and redrawn. This process is continued untilappropriate channel diameters and the desired number of array elementsare achieved. By adjusting the ratio of the diameter of the etchableglass rod to that of the outside dimension of the inert glass tubing,the center-to-center spacing of the rods and their diameters in thefinished product become independently adjustable parameters.

Once the fabrication process is complete, the NCG material is waferedperpendicular to the direction of the channels with a diamond saw andthen polished to produce 0.1–1.0 mm sections of material. The channelglass of the array structure is then etched away with an acid solution.

A hexagonal close packing arrangement of channel glasses, after acidetching, contains typically 10⁷ channels and is uniform throughout. Thechannel diameter is typically 450 nm and the center-to-center spacing isapproximately 750 nm. The type of array structure described above isuseful in the NCG array hybridization assembly in accordance with thepresent invention. In this configuration, the tapered sample wellstructure defines each group of channels serving as a specifichybridization test site.

A second type of hexagonal array structure, in which separated clustersof channels are formed during the fabrication process, exhibits an openarray structure with typical channel diameters of 300 nm. The overallglass structure consists of an array of 18 μm diameter subarrays, eachserving to contain a specific DNA probe or target, and spaced typically25 μm apart from neighboring arrays.

EXAMPLE 2 Well Arrays Defining Discrete and Isolated Binding Regions

The NCG hybridization arrays described in Example 1 are bonded on theupper side to a polymeric layer containing an array of orifices whichalign with the array of nanochannel bundles and serve as sample wellsfor placement of a substantially homogeneous sample of a biomolecule(e.g., a single DNA species) within each hybridization site. Thispolymeric sample well array also provides physical support to thefragile NCG wafer.

The polymeric array of orifices are fabricated using methods known inthe art. For example, this polymeric layer suitable for use herein canbe obtained from MicroFab Technologies, Inc. The orifices are fabricatedusing excimer laser machining. This method is preferred because existingtechnology is employed allowing for low cost/high volume manufacturing,as is currently being done in the microelectronics industry.

Development of the polymeric array comprises four task: (1) materialsselection; (2) ablation tooling and process development; (3) laminationtooling and process development; and (4) production of high density andultra-high density polymeric arrays. These tasks are undertaken asfollows:

Part A: Materials Selection.

The materials useful in the polymeric array are filled polymers, epoxyresins and related composite (e.g., “circuit-board”-type) materials.Because it is a standard process in the microelectronics industry, thepresent invention most advantageously employs polymeric materials withthe adhesive applied by the commercial vendor of the material forexample, a polyamide with a 12 μm thick layer of a B-stage (heat curing)adhesive

The primary requirements for the polymeric array material to be usedare:

1. High Suitability for Excimer Laser Machinability:

i. high absorption in UV (e.g., >4×10⁵/cm at 193 nm),

ii. high laser etch rate (e.g., 0.5 μm/pulse ) and low hole taper(reduction in hole diameter with depth into material, e.g., <3°);

2. Obtainable in thicknesses up to 1 mm;

3. Obtainable with B-stage adhesive on one side which is both laserablatable and suitable for bonding to the nanoporous wafer;

4. High rigidity and thermal stability (to maintain accurate alignmentof samplewell and NCG wafer features during lamination);

5. Compatibility with DNA solutions (i.e., low nonspecific binding)

Part B: Ablation Tooling and Processing

Contact mask excimer laser machining is a preferred processing techniquebecause it is a lower cost technique than projection mask excimer lasermachining. A projection mask is, however, employed when the feature sizeless than 50 μm. One or more masks with a variety of pattern sizes andshapes are fabricated, along with fixtures to hold the mask and materialto be ablated. These masks are employed to determine the optimalmaterial for laser machining and the optimal machining conditions (i.e.,mask hole size, energy density, input rate, etc.). Scanning electronmicroscopy and optical microscopy are used to inspect the excimer lasermachined parts, and to quantify the dimensions obtained, including thevariation in the dimensions.

In addition to ablating the sample wells into the polymeric material,the adhesive material is also ablated. This second ablation isundertaken so that the diameter of the hole in the adhesive is madelarger than diameter of the sample well on the adhesive side of thepolymeric material. This prevents the adhesive from spreading into thesample well and/or the nanoporous glass during lamination.

Part C: Lamination Tooling and Processing

Initial lamination process development is carried out using unablatedpolymeric material (or alternatively, using glass slides and/or siliconwafers). Cure temperature, pressure, and fixturing are optimized duringthis process development. Thereafter, the optimized processingparameters are employed to laminate both nanoporous wafers and polymericarrays. The final lamination is done such that the alignment of the twolayers creates functional wells.

Part D: Production of Polymeric Arrays

The optimal mask patterns and excimer laser parameters are determinedand thereafter employed in the manufacture of contact masks and materialholding fixtures. Typically, fabrication is done so as to produce alarge number (>100) of parts as the masks wear out with use).

EXAMPLE 3 Porous Silicon Wafers

Two general types of porous silicon devices are prepared according tothe process described herein. First, known microfabrication methods areused to fabricate wafers, bounded by integral sample wells. Second,uniformity porous wafer structures are bonded to the same orifice samplewell arrays that were described previously (Example 2) for NCG glassarrays. Porous silicon designs are advantageously employed hereinbecause of their adaptability to low cost mass production processes andtheir ability to incorporate in the fabrication process structuralelements that function in fluidic entry and exit from the hybridizationsite and structures (e.g., electrodes) that may function inhybridization detection. Stable, open-cell porous materials are used toaccomplish enhancements and to introduce qualitatively new features inthese devices, whereby the surface area of discrete and isolated bindingregions is increased by a factor of 100 to 1000 in hybridization-basedelectronic, fluorescence and radiation-based DNA detectors. Inaccomplishing this objective, controlled introduction ofhigh-surface-area supports at the surface detection site is employed.

Thin-film processing technology is used to deposit chemically inert andthermally stable microporous materials. Materials and processing methodsare selected to achieve low-cost semiconductor batch fabrication ofintegrated semiconductor detectors. The microchip device provides insitu multisite analysis of binding strength as ambient conditions arevaried. Porous silicon materials are fabricated in oriented, pore arraysor random interconnected networks and with pore diameters selected overthe range from 2 nm to several micrometers.

Porous silicon is produced most easily through electrochemical etching.It can be processed into two important pore structures, interconnectednetworks and oriented arrays. The pore diameter is tailored fromapproximately 2 nm to micrometer dimensions by selection of doping andelectrochemical conditions. For n-type material, etching is thought toproceed through a tunneling mechanism in which electrons are injectedinto the pore surface through field concentration effects. In the caseof p-type material the mechanism seems to be through moderation ofcarrier supply at the electrolyte/silicon interface. In practice, thefollowing structures can be fabricated:

i) a dense interconnected network layer with porosity of 40–60% andsilicon filament size in the nanometer size regime. This is most easilyobtained in lightly doped (<1 Ω-cm resistivity) p-type silicon.

ii) a interconnected branched network of pores of typically 10-nmdiameter, axis preferentially oriented along <100> direction, andporosity of 30–80% depending on etching conditions. This is obtained inp-type material of 10⁻¹ to 10⁻² Ω-cm resistivity.

iii) dense oriented arrays of pores oriented with axis along <100 >direction and with pore diameters in the range of 10 to 100 nm. Obtainedin p-type material with resistivity less than 10⁻² Ω-cm.

iv) dense oriented arrays of pores oriented along <100> direction andwith pore diameters in the range less than 10 nm. Obtained in n-typematerial with resistivity between 10⁻¹ and 10⁻² Ω-cm.

v) dense oriented arrays of rectangular pores oriented with axis along<100 > direction, rectangle side defined by {001} planes, and with porediameters in the range less than 100 nm. Obtained in p-type materialwith resistivity between 10⁻¹ and 10⁻² Ω-cm.

vi) low density interconnected networks of large (1-μm-diameter) pores.This occurs in lightly doped n-type material.

These materials are fabricated on the device structures described above.

Characterization can be undertaken by scanning electron microscopy. Thesurface wetting properties are varied using vapor treatment withsilylation materials and chlorocarbons.

High-porosity dielectrics which function as molecular sieves areproduced by nuclear track etching. While nuclear track etching is usedto produce these molecular sieves in a wide range of inorganicmaterials, it is most often used with dielectrics such as mica andsapphire. In this method, described in U.S. Pat. No. 3,303,085 (Price,et al.), a substrate is first bombarded with nuclear particles(typically several MeV alpha particles) to produce disturbances or“tracks” within the normal lattice structure of the material and thenwet-etched to produce pores which follow the tracks caused by thenuclear particles. More specifically, Price et al. disclose that theexposure of a mica substrate to heavy, energetic charged particles willresult in the formation of a plurality of substantially straight tracksin its lattice structure and that these tracks can be converted intopores by wet etching the substrate.

Pore sizes and overall porosity are variably controllable with porestypically 0.2 μm in diameter and densities on the order of 10⁹/cm².Particle track depths are energy dependent on the incident particlebeam, but resulting pores can be extended through an entire 500-μm-thicksubstrate. Incorporation of these materials on the device structuresshown above is readily accomplished. In addition, the use ofimplantation-etched dielectrics as the sensor element has advantagesversus the porous silicon approach since the material is hydrophilic.

A preferred device is the porous silicon array wafer with integralsample wells illustrated in FIG. 3. This may be constructed as follows:A four inch diameter, 100 μm thick wafer of crystalline silicon (n-type,doped with 10¹⁵ P/cm³) with axis oriented along <100> direction iscoated with photoresist and exposed to light through a mask to define a50×50 array of 200 μm square areas having 200 μm space between themacross the 2 cm×2cm central area of the wafer. The process described byV. Lehmann (J. Electrochem. Soc. 140(100):2836–2843 (1993)) is then usedto create patches of closely spaced pores of diameter 2–5 μm, orientedperpendicular to the wafer surface, within each square area defined inthe photolithographic step. A 300 μm thick wafer of silicon dioxide iscoated with photoresist and exposed to light through the same mask usedto define 200 μm square porous regions in the silicon wafer, and acidetching is conducted to create 200 μm square holes in the silicondioxide wafer. The silicon dioxide wafer is then aligned with andlaminated to the porous silicon wafer using a standard wafer bondingprocess to form the integral structure shown in the figure. During thehigh temperature annealing step, the silicon surface of each pore isoxidized to form a layer of silicon dioxide. The epoxysilane-aminelinkage procedure described in EXAMPLE 4 is then carried out tocovalently attach amine-containing biopolymer species to the walls ofthe pores.

EXAMPLE 4 Oligonucleotide Attachment to Glass/SiO₂

Part A: Epoxysilane Treatment of Glass

A stock solution of epoxysilane is freshly prepared with the followingproportions: 4 ml 3-glycidoxypropyl-trimethoxysilane, 12 ml xylene, 0.5ml N,N-diisopropylethylamine (Hunig's base). This solution is flowedinto the pores of the wafer, then the wafer is soaked for 5 hours in thesolution at 80° C., then flushed with tetrahydrofuran, dried at 80° C.,and placed in a vacuum desiccator over Drierrite™ stored in a desiccatorunder dry argon.

Part B: Attachment of Oligonucleotide

Oligonucleotide, bearing 5′- or 3′-alkylamine (introduced during thechemical synthesis) is dissolved at 10 μM–50 μM in water and flowed intothe porous silica wafer. After reaction at 65° C. overnight the surfaceis briefly flushed with water at 65° C., then with 10 mM triethylamineto cap off the unreacted epoxy groups on the surface, then flushed againwith water at 65° C. and air dried. As an alternative to attachment inwater, amine-derivatized oligonucleotides can be attached toepoxysilane-derivatized glass in dilute (eg., 10 mM–50 mM) KOH at 37° C.for several hours, although a higher background of nonspecific bindingof target sample DNA to the surface (independent of base pairing) mayoccur during hybridization reaction.

EXAMPLE 5 Robotic Fluid Delivery

A Hamilton Microlab 2200™robotic fluid delivery system, equipped withspecial low volume syringes and 8-position fluid heads, capable ofdelivering volumes of 10–100 nl at 500 μm xyz stepping and a few percentprecision. Using this equipment 40-nl samples of biomolecules (e.g.,DNA, olgionucleotides and the like) are placed into the wells of thehigh density NCG wafer. A piezoelectrically controlled substage customfitted for the Microlab 2200 permits xy positioning down to submicronresolution. For 1-nl samples, custom fabricated needles are employed.The eight-needle linear fluid head is operated in staggered repetitivesteps to generate the desired close spacing across the wafer. The systemhas a large stage area and rapid motion control, providing the capacityto produce hundreds of replicate hybridization wafers.

Part A: Microfab Microfluidic Jets

Methods are known in the art (Microfab Technologies, Inc.) fordelivering sub-nanoliter microdroplets of fluids to a surface atsubmicron precision. A microjet system capable of deliveringsubnanoliter DNA solutions to the wafer surface is employed as follows:For placement of DNA into individual hybridization sites withinultra-high density wafers, with volumes of one nl (corresponding to a130 μm sphere or 100 μm cube) commercially available dispensingequipment using ink-jet technology as the microdispensing method forfluid volume below is employed. The droplets produced using ink-jettechnology are highly reproducible and can be controlled so that adroplet may be placed on a specific location at a specific timeaccording to digitally stored image data. Typical droplet diameters fordemand mode ink-jet devices are 30–100 μm, which translates to dropletvolumes of 14–520 pl. Droplet creation rates for demand mode ink-jetdevices are typically 2000–5000 droplets per second. Thus, both theresolution and throughput of demand mode inkjet microdispensing are inthe ranges required for the ultrahigh density hybridization wafer.

Part B: Microdispensing System

The microdispensing system is modified from a MicroFab drop-on-demandink-jet type device, hereafter called a MicroJet device such that thistype of device can produce 50 μm diameter droplets at a rate of 2000 persecond. The operating principles of this type of device are known (D. B.Wallace, “A Method of Characteristics Model of a Drop-On-Demand Ink-JetDevice Using an Integral Drop Formation Method,” ASME publication89-WA/FE4, December 1989) and used to effect the modification. Toincrease throughput, eight of these devices are integrated into a linearray less than 1 inch (25 mm) long. The eight devices are loaded withreagent simultaneously, dispense sequentially, and flush simultaneously.This protocol is repeated until all of the reagents are dispensed. Mostof the cycle time is associated with loading and flushing reagents,limiting the advantages of a complex of parallel dispensing capability.Typical cycle time required is as on the following order: 1 minute forflush and load of 8 reagents; 30 seconds to calibrate the landinglocation of each reagent; 15 seconds to dispense each reagent on onelocation of each of the 16 genosensors, or 2 minutes to dispense all 8reagents. Total time to load and dispense 8 reagents onto 16 sensors isthus 3.5 minutes. Total time for 64 reagents onto 16 sensors would be 28minutes. The microdispensing system will consist of the subsystemslisted below:

A. MicroJet Dispense Head—An assembly of 8 MicroJet devices and therequired drive electronics. The system cost and complexity are minimizedby using a single channel of drive electronics to multiplex the 8dispensing devices. Drive waveform requirements for each individualdevice are downloaded from the system controller. The drive electronicsare constructed using conventional methods.

B. Fluid Delivery System—A Beckman Biomec is modified to act as themultiple reagent input system. Between it and the MicroJet dispense headare a system of solenoid valves, controlled by the system controller.They provide pressurized flushing fluid (deionized water or saline) andair to purge reagent from the system and vacuum to load reagent into thesystem.

C. X-Y Positioning System—A commercially available precision X-Ypositioning system, with controller, is used. Resolution of 0.2 μm andaccuracy of 2 μm are readily obtainable. The positioning system is sizedto accommodate 16 sensors, but MicroJet dispense head size, purgestation, and the calibration station represent the main factors indetermining overall size requirements.

D. Vision System—A vision system is used to calibrate the “landing zone”of each MicroJet device relative to the positioning system. Calibrationoccurs after each reagent loading cycle. Also, the vision system locateseach dispensing site on each sensor when the 16 sensor tray is firstloaded via fiducial marks on the sensors. For economy, a software basedsystem is used, although a hardware based vision system can beadvantageously employed.

E. System Controller—A standard PC is used as the overall systemcontroller. The vision system image capture and processing also resideon the system controller.

EXAMPLE 6 Liquid Flow-Through

In order to bind DNA probes or targets within the pores of themicrofabricated hybridization support, carry out the hybridization andwashing steps, process the material for re-use, and potentially recoverbound materials for further analysis, a means is provided for flow ofliquids through the wafer. To enable flow of liquid through thehybridization wafer, it is packaged within a 2 mm×4 mm polypropyleneframe, which serves as an upper reservoir and structure for handling. Apolypropylene vacuum chamber with a Deltin o-ring around its upper edgeto permit clamping of the wafer onto the vacuum manifold to form a sealis employed. The vacuum assembly is illustrated in FIG. 4. For controlof fluid flow through the wafer a screw-drive device with feedbackcontrol is provided.

EXAMPLE 7 Synthesis and Derivatization of Oligonucleotides

Oligonucleotides to be used in the present invention are synthesized bythe phosphoramidite chemistry (Beaucage, S. L. and Caruthers, M. H.1981. Tet. Lett. 22:1859–1862) using the segmented synthesis strategythat is capable of producing over a hundred oligonucleotidessimultaneously (Beattie, K. L., Logsdon, N. J., Anderson, R. S.,Espinosa-Lara, J. M., Maldonado-Rodriguez, R. and Frost, J. D. III.1988. Biotechnol. Appl. Biochem. 10:510–521; Beattie, K. L. and Fowler,R. F. 1991. Nature 352:548–54926,27). The oligonucleotides can bederivatized with the alkylamino function during the chemical synthesis,either at the 5′-end or the 3′-end.

Part A: Chemistry of Attachment to Glass

Optimal procedures for attachment of DNA to silicon dioxide surfaces arebased on well-established silicon chemistry (Parkam, M. E. and Loudon,G. M. (1978) Biochem. Biophys. Res. Commun., 1: 1–6; Lund, V., Schmid,R., Rickwood, D. and Hornes, E. (1988) Nucl. Acids Res. 16:10861–10880). This chemistry is used to introduce a linker group ontothe glass which bears a terminal epoxide moiety that specifically reactswith a terminal primary amine group on the oligonucleotides. Thisversatile approach (using epoxy silane) is inexpensive and provides adense array of monolayers that can be readily coupled to terminallymodified (amino- or thiol-derivatized) oligonucleotides. The density ofprobe attachment is controlled over a wide range by mixing long chainamino alcohols with the amine-derivatized oligonucleotides duringattachment to epoxysilanized glass. This strategy essentially produces amonolayer of tethered DNA, interspersed with shorter chain alcohols,resulting in attachment of oligonucleotides down to 50 Å apart on thesurface. Variable length spacers are optionally introduced onto the endsof the oligonucleotides, by incorporation of triethylene glycolphosphoryl units during the chemical synthesis. These variable linkerarms are useful for determining how far from the surface oligonucleotideprobes should be separated to be readily accessible for pairing with thetarget DNA strands. Thiol chemistry, adapted from the method ofWhitesides and coworkers on the generation of monolayers on goldsurfaces (Randall lee, T., Laibinis, P. E., Folkers, J. P. andWhitesides, G. M. (1991) Pure & Appl. Chem. 63: 821–828 and referencescited therein.), is used for attachment of DNA to gold and platinumsurfaces. Dithiols (e.g., 1,10-decanedithiol) provide a terminal,reactive thiol moiety for reaction with bromoacetylatedoligonucleotides. The density of attachment of DNA to gold or platiniumsurfaces is controlled at the surface-activation stage, by use ofdefined mixtures of mono- and dithiols.

Part B: Surface Immobilization of Recombinant Vector DNA , cDNA and PCRFragments

The chemical procedures described above are used most advantageously forcovalent attachment of synthetic oligonucleotides to surfaces. Forattachment of longer chain nucleic acid strands to epoxysilanized glasssurfaces, the relatively slow reaction of surface epoxy groups with ringnitrogens and exocylic amino groups along the long DNA strands isemployed to achieve immobilization. Through routine experimentation,optimal conditions for immobilization of unmodified nucleic acidmolecules at a few sites per target are defined, such that the bulk ofthe immobilized sequence remains available for hybridization. In thecase of immobilization tonanochannels coated with platinum or gold,hexylamine groups are first incorporated into the target DNA usingpolymerization (PCR or random priming) in the presence of5-hexylamine-dUTP, then a bromoacetylation step is carried out toactivate the DNA for attachment to thiolated metal surfaces. Again,routine experimentation is employed (varying the dTTP/5-hexylamine-dUTPratio and the attachment time) to define conditions that givereproducible hybridization results.

The foregoing procedure (omitting the bromoacetylation step) can alsoserve as an alternative method for immobilization of target DNA to glasssurfaces.

Part C: DNA Binding Capacity

Based upon quantitative measurements of the attachment of labeledoligonucleotides to flat glass and gold surfaces, the end attachmentplaces the probes 50–100 Å apart on the surface, corresponding to up to10⁸ probes in a 50 μm×50 μm area. Approximately 10¹⁰–10¹¹oligonucleotide probes can be tethered within a 50 μm cube of poroussilicon in the nanofabricated wafer. The density of boundoligonucleotides per cross sectional area is estimated by end-labelingprior to the attachment reaction, then quantitating the radioactivityusing the phosphorimager. Known quantities of labeled oligonucleotidesdried onto the surface are used to calibrate the measurements of bindingdensity. From data on the covalent binding of hexylamine-bearing plasmidDNA to epoxysilanized flat glass surfaces in mild base, it is known thatat least 10⁷ pBR322 molecules can be attached per mm² of glass surface.Based on this density within the pores of the nanofabricated wafer,immobilization of 10⁹–10¹⁰ molecules of denatured plasmid DNA per mm² ofwafer cross section are achieved.

EXAMPLE 8 Hybridization Conditions

Part A: Sample Preparation

The target DNA (analyte) is prepared by the polymerase chain reaction,incorporating [³²P]nucleotides into the product during the amplificationor by using gamma-³²P[ATP]+ polynucleotide kinase to 5′-label theamplification product. Unincorporated label is removed by Centriconfiltration. Preferably, one of the PCR fragments is 5′-biotin-labeled toenable preparation of single strands by streptavidin affinitychromatography. The target DNA is dissolved in hybridization buffer (50mM Tris-HCl, pH 8, 2 mM EDTA, 3.3M tetramethylammonium chloride) at aconcentration of at least 5 nM (5 fmol/μl) and specific activity of atleast 5,000 cpm/fmol. PCR fragments of a few hundred bases in length aresuitable for hybridization with surface-tethered oligonucleotides of atleast octamer length.

Part B: Hybridization.

The target DNA sample is flowed into the porous regions of the chip andincubated at 6° C. for 5–15 minutes, then washed by flowinghybridization solution through the porous chip at 18° C. for a similartime. Alternatively, hybridization can be carried out in buffercontaining 1M KCL or NaCl or 5.2M Betaine, in place oftetramethylammonium chloride.

Part C: Optimization of Hybridization Selectivity (DiscriminationAgainst Mismatch-Containing Hybrids

Although the experimental conditions described above generally yieldacceptable discrimination between perfect hybrids andmismatch-containing hybrids, some optimization of conditions may bedesirable for certain analyses. For example, the temperature ofhybridization and washing can be varied over the range 5° C. to 30° C.for hybridization with short oligonucleotides. Higher temperatures maybe desired for hybridization using longer probes.

EXAMPLE 9 Quantitative Detection of Hybridization

Part A: Phosphorimager and Film Detection

The detection and quantitation of hybridization intensities is carriedout using methods that are widely available: phosphorimager and film.The Biorad phosphorimager has a sample resolution of about 100 μm and iscapable of registering both beta emission and light emission fromchemiluminescent tags. Reagent kits for chemiluminescence detectionavailable from Millipore and New England Nuclear, which produce light of477 and 428 nm, respectively, are advantageously used with the Bioradinstrument. Chemiluminescent tags are introduced into the target DNAsamples (random-primed vector DNA or PCR fragments) using the proceduresrecommended by the supplier. Thereafter, the DNA is hybridized to thenanoporous wafers bearing oligonucleotide probes. Radioactive tags (³²Pand ³³P, incorporated by random priming and PCR reaction) are also usedin these experiments. Film exposure is used for comparison. In the caseof hybridization of labeled oligonucleotides with surface-immobilizedtarget DNAs, most preferably the radioactive tags (incorporated usingpolynucleotide kinase) are used, since optimal chemiluminescent taggingprocedures for oligonucleotides are generally not available.

Part B: CCD Detection Devices

CCD genosensor devices are capable of maximum resolution and sensitivityand are used with chemiluminescent, fluorescent and radioactive tags(Lamture, J. L., Varma, R., Fowler, R., Smith, S., Hogan, M., Beattie,K. L., Eggers, M., Ehrlick, D., Hollis, M. and Kosicki, B. 1993. Nature,submitted).

EXAMPLE 10 Genosensor Experiment; Mutation Detection in Exon 7/8 Regionof Hamster hprt Gene

The hprt gene is used extensively as a model system for studies ofmutation. The gene has been cloned and sequenced from several mammals. Avariety of mutations in this gene are known and were characterized byDNA sequencing, in the hamster (induced by chemicals and radiation inChinese Hamster Ovary cell lines) and from humans (associated with LeschNyhan syndrome). A significant fraction of hprt mutations are found in ashort region of the gene encoded by exons 7 and 8. The nucleotidesequence of the normal and mutant genes are found in the followingreferences: Edwards, A., Voss, H., Rice, P., Civitello, A., Stegemann,J., Schwager, C., Zinimermann, J., Erfle, H., Caskey, C. T. and Ansorge,W. (1990), Automated DNA Sequencing of the Human HPRT Locus, Genomics,6:593–608; Gibbs, R., Nguyen, P.-N., Edwards, A., Civitello, A. andCaskey, C. T. (1990), Multiplex DNA Deletion Detection and ExonSequencing of the Hypoxanthine Phosphoribosyltransferase Gene inLesch-Nyhan Families, Genomics, 7:235–244; Yu, Y., Xu, Z, Gibbs, R. andHsie, A. (1992), Polymerase chain reaction-based Comprehensive Procedurefor the Analysis of the Mutation Spectrum at the Hypoxanthine-guaninePhosphoribosyltransferase locus in Chinese Hamster Cells, Environ. Mol.Mutagen., 19:267–273; and Xu, Z., Yu, Y., Gibbs, R., Caskey, C. T. andHsie, A. (1993), Multiplex DNA Amplification and Solid-phase DirectSequencing at the hprt Locus in Chinese Hamster Cells, Mutat. Res.,282:237–248. The nucleotide sequence of the cDNA of hamster hprt exon7/8 region is listed as follows:

(SEQ ID NO: 1)                   500                   520                  540 GCAAGCTTGC TGGTGAAAAG GACCTCTCGA AGTGTTGGATATAGGCCAGA CTTTGTTGGA                   560                   580                  600 TTTGAAATTC CAGACAAGTT TGTTGTTGGA TATGCCCTTGACTATAATGA GTACTTCAGG GATTTGAATC

The following represents the nucleotide sequence of hamster hprt genomicDNA in the exon 7/8 region where the CHO mutations are depicted above(l) and the human (h) and mouse (m) sequence differences below (l). TheDNA sequence which begins with “5′-aacagCTTG” and which ends with“5′-GACTgtaag” is designated as SEQ ID NO:2 for sequences of hamster,human and mouse and SEQ ID NO:3 for the sequence of CHO cells. Theremaining DNA, beginning with “5′-tacagTTGT” and ending with “GAATgtaat”is designated as SEQ ID NO:4 for sequences of hamster, human and mouseand SEQ ID NO:5 the sequence of CHO cells.

                             ----------                                  ↑-aacagCTTGCTGGTGAAAAGGACCTCTCGAAGTGTTGGATATAGGCCAG                         ↓  ↓             ↓ ↓                          C A             C A                          h  h             m h                             G        -                             ↑        ↑ACTgtaag----tacagTTGTTGGATTTGAAATTCCAGACAAGTTTGTTG                    +A             C                     ↑             ↑TTGGATATGCCCTTGACTATAATGAGTACTTCAGGATTTGAATgtaat- ↓                       ↓         ↓ A                       A         A h                       h         h

The small letters in the beginning of the sequence represent intronsequence on the 5′-side of exon 7. Some flanking intron sequence betweenexons 7 and 8 is shown (in small letters) on the second line, and at theend there is again a small stretch of intron sequence following exon 8.Underlined bases in the sequence represent mutations for which DNAsamples are available, which can be used to demonstrate that a DNA chiptargeted to this region can detect and identify mutations. Above thesequences are displayed mutations in hamster (CHO) cells induced bychemicals and radiation, including a 10-base deletion (top line), singlebase deletion (second line), single base insertion (third line) andsingle base substitutions (second and third lines). Below the sequencesare shown single base differences between hamster and human (h) andmouse (m).

The set of oligonucleotide probes (of 8 mer–10 mer in length)overlapping by two bases across the exon 7/8 region is depicted belowfor SEQ ID Nos:2–5:

          ----2----     ----4----     ----6----   ----1----     ----3----     ----5----     --7---aacagCTTGCTGGTGAAAAGGACCTCTCGAAGTGTTGGATATAGGCCAG                         ↓  ↓     ↓       ↓ ↓                         C  A    -10      C A               ----8-----      ----10----     -12--7-                    ----9-----      ----11---ACTgtaag----tacagTTGTTGGATTTGAAATTCCAGACAAGTTTGTTG                             ↓        ↓                             G        ---12-     ----14---     ----16----     ----18---   ----13---     ----15---      ----17---TTGGATATGCCCTTGACTATAATGAGTACTTCAGGGATTTGAATgtaat ↓                   ↓   ↓         ↓ A                  +A   A         A                                   C

This set of probes is selected to detect any of the mutations in thisregion, and the lengths are adjusted to compensate for base compositioneffects on in duplex stability (longer probes for AT-rich regions). Thesequences of probes and primers are given in Table I, as follows:

TABLE I OLIGONUCLEOTIDES FOR hprt MUTATION DETECTION PCR primers forexons 7 & 8: Name Sequence (5-3) MHEX71 GTTCTATTGTCTTTCCCATATGTC (SEQ IDNO:6) MHEX82 TCAGTCTGGTCAAATGACGAGGTGC (SEQ ID NO:7) HEX81CTGTGATTCTTTACAGTTGTTGGA (SEQ ID NO:8) HEX82 CATTAATTACATTCAAATCCCTGAAG(SEQ ID NO:9) 9mer with amine at 5′-end: Name Sequence (5′–>3′) −A (554)TGCTGGAAT +A (586/7) ACTCATTTATA (SEQ ID NO: 10) −10 (509–518)TATATGAGAG (SEQ ID NO: 11) A–G (545) ATTCCAAATC (SEQ ID NO: 12) G–C(601) CAAATGCCT 1 AGCAAGCTG 2 TTTCACCAG 3 AGGTCCTTT 4 CTTCGAGAG 5TCCAACACT 6 GCCTATATC 7 AGTCTGGC 8 TCCAACAACT (SEQ ID NO:13) 9ATTTCAAATC (SEQ ID NO: 14) 10 GTCTGGAAT 11 ACAAACTTGT (SEQ ID NO: 15) 12TCCAACAAC 13 GGGCATATC 14 TAGTCAAGG 15 ACTCATTATA (SEQ ID NO: 16) 16CTGAAGTAC 17 CAAATCCCT 18 AATTACATTCA (SEQ ID NO: 17)

A high-density or ultra-high density microfabricated device according tothe above examples is constructed and attachment of oligonucleotideprobes is carried out within the bounded regions of the wafer. Includedare the normal probes (1–18) plus the specific probes that correspond tofive different known mutations, including the above mutations (sites 19and 20, respectively). The foregoing uses two sets of PCR primers (TableI) to amplify the exons 7/8 region of hamster genomic DNA. A radioactivelabel (³²P) is incorporated into the PCR fragments during amplification,which enables detection of hybridization by autoradiography orphosphorimager. FIG. 5 illustrates the results when the above probes areattached at one end to the surface at specific test sites within the DNAchip (numbered as above). Idealized hybridization patterns for two ofthe mutants (10-base deletion on left and A–G transition on right) areshown at the bottom.

EXAMPLE 11 Profiling of Gene Expression Using cDNA Clones Arrayed inPorous Silicon

Part A: Fabrication of Porous Silicon Wafer

The procedure outlined in EXAMPLE 3 for fabrication of a porous siliconwafer with integral sample wells is followed, to yield a wafer with a50×50 array of 200 μm square patches of pores, spaced 400 μm apart(center-to-center) over the surface of the wafer. The pores of the waferare activated to bind amine-derivatized polynucleotides by reaction withepoxysilane, as described in EXAMPLE 4.

Part B: Formation of cDNA Array

A set of 2,500 M13 clones, selected from a normalized human cDNAlibrary, is subjected to the polymerase chain reaction (PCR) in thepresence of 5′-hexylamine-dUTP to amplify the cDNA inserts andincorporate primary amines into the strands. The PCR products areethanol-precipitated, dissolved in water or 10 mM KOH, heat-denatured at100° C. for 5 min., then quenched on ice and applied to individualsample wells of the porous wafer suing a Hamilton Microlab 2200 fluiddelivery system equipped with an 8-needle dispensing head. After allcDNA fragments are dispensed, a slight vacuum is briefly applied frombelow to ensure that fluid has occupied the pores. Following incubationat room temperature overnight or at 60° C. for 30–60 minutes, the porouswafer is flushed with warm water, then reacted with 50 mM triethylamineto cap off the unreacted epoxy groups on the surface, then flushed againwith warm water and air dried.

Part C: Preparation of Labeled PCR Fragments Representing the 3′-regionsof Expressed Genes

Cytoplasmic RNA is extracted from cultured cells by the method ofChomczynski and Sacchi (Anal. Biochem.162:156–159 (1993)), treated withDNAse I to remove DNA contamination, then extracted withphenol/chloroform and ethanol precipitated. Reverse transcriptions andPCR are performed as described in the “differential display” protocol ofNishio et al. (FASEB J. 8:103–106 (1994)). Prior to hybridization, PCRproducts are labeled by random priming in the presence of [A-³²P]dNTPs,and unincorporated label is removed by Centricon filtration.

Part D: Hybridization of Expressed Sequences to cDNA Array

Prior to hybridization, a solution of 1% “Blotto” or 50 mMtripolyphosphate is flowed through the porous silicon wafer to minimizethe nonspecific binding of target DNA, then the porous silicon array iswashed with hybridization solution (50 mM Tris-HCl, pH 7.5, 1 mM EDTA,1M NaCl). Labeled PCR fragments representing the 3′-end of expressedgenes are recovered from the Centricon filtration units in hybridizationbuffer, and the entire porous wafer is flooded with this DNA solution.The porous hybridization module is placed at 65° C. and a peristalticpump, connected to the lower vacuum chamber, is used to gradually flowthe labeled DNA through the pores of the wafer over the course of 30–60minutes. The porous wafer is washed three times with hybridizationbuffer at 65° C.

Part E: Quantitation of Hybridization Signals

Following hybridization and washing, the porous wafer is briefly dried,then placed onto the phosphor screen of a phosphorimager and kept in thedark for a period of time determined by the intensity of label. Thephosphor screen is then placed into the phosphorimager reader forquantitation of individual hybridization signals arising from eachporous region in the array.

FIG. 6 illustrates results obtainable from a hybridization experiment.Total cytoplasmic mRNA is isolated from cells cultured under twoconditions and subjected to the “differential display” proceduredescribed above to prepare fragments representative of individual mRNAspecies present under the two conditions. These samples are hybridizedto two identical cDNA arrays, to yield the two hybridization signalpatterns shown. These patterns represent the profile of expressed genesunder the two different culture conditions (for example in the presenceand absence of a drug or chemical that induces a change in theexpression of some genes). Note that overall, the pattern ofhybridization is similar for the two conditions, but as expected for adiffential expression of certain genes under the two conditions, thereare a few hybridization signals that are seen only for culture condition1 and a few that are seen only for culture condition 2. The box in thelower left, reproduced at the bottom of the figure to assist visualcomparison, represents several differences in the gene expressionprofile. The squares represent sites where hybridization has occurredand the darkness of the squares is proportional to the number of labeledfragments present at each site.

1. A device comprising a substrate containing a multiplicity of samplewells, wherein each of said sample wells contains discrete and isolatedregions arrayed within said sample well, and wherein each of saiddiscrete and isolated regions is a microchannel and contains ahomogeneous sample of polynucleic acids fixed thereto.
 2. The device ofclaim 1, wherein said substrate is fabricated from glass.
 3. The deviceof claim 1, wherein said sample well is tapered.
 4. The device of claim1, wherein said device is fabricated from porous silicon.
 5. The deviceof claim 1, wherein the device is microfabricated.
 6. A devicecomprising a plurality of genosensors, wherein each genosensor iscontained in a sample well.
 7. The device of claim 6, wherein saiddevice is fabricated from glass.
 8. The device of claim 6, wherein saidsample well is tapered.
 9. The device of claim 6, wherein saidgenosensor contains flowthrough channels.
 10. The device of claim 6,comprising at least 9 sample wells.
 11. The device of claim 6,comprising at least 10 sample wells.
 12. The device of claim 6,comprising at least 30 sample wells.
 13. The device of claim 6, whereinsaid device is fabricated from porous silicon.
 14. The device of claim6, wherein the device is microfabricated.